Oils and fats have been important components of human diet from very ancient times. They are considered as sources of energy, essential fatty acids and fat soluble vitamins and have key role in metabolism. Today it has been proved that some fats and oils have also functional properties in human body and positive impact on human health. These compounds are known as functional, nutritional, medical or pharmaceutical lipids. According to differences in stages of digestion, absorption and metabolism of diacylglycerol oil (DAG oil) in body and its favorable effects in preventing many diseases, it seems that this oil is a good option for production as functional oil. Diacylglycerols (DAGs) are esters of glycerol with two fatty acids (FFAs) which have two structural isomers, i.e., sn-1,2(2,3) DAG and sn-1,3 DAG. By definition, DAG oil is an oil containing at least 10% DAG whereas commercial one contains 80% DAG, maximum 20% triacylglycerol (TAG) and 5% monoacylglycerol (MAG). In 1999, for the first time, this oil was produced from soybean and canola oils in Japan (Econa oil). In 2000 and 2003, this oil was registered as a GRAS (Generally Recognized As Safe) compound by the FDA (Food and Drug Administration) in USA. Also in both countries, DAG oil referred as functional cooking oil. DAG oil can be produced with various routes such as hydrolysis, glycerolysis and esterification or combination of these methods. Each of these production methods can be done with alkaline inorganic chemical or enzyme catalysts. In this study DAG oil was produced from safflower oil by enzymatic glycerolysis in solvent-free reaction system. Lipozyme TL IM was used as biocatalyst which is an immobilized 1,3-position-specific lipase from Thermomyces lanuginosa. Different variables, including temperature, time, mole ratio of glycerol to oil and enzyme percentage were considered in oil production. Finally, optimization of the reaction and determination of optimum levels for each variable was done using response surface methodology (RSM). The results showed that there was no significant difference between the amount of DAG oil predicted by the software and its actual content and the model could well be used for production of desired amount of DAG. Optimal conditions were found to be 0.75 % enzyme, 5.3g glycerol, temperature of 46.9 ° C and reaction time of 4 h. Finally, physicochemical properties of DAG oil and safflower oil (raw material) were compared and results suggested that despite of slight differences in some properties, DAG oil could be used as a replacement for safflower oil and even other conventional oils in food industry